Synthesis, antimicrobial activity and gene structure of a novel member of the dermaseptin B family.

Dermaseptins are a family of cationic (Lys-rich) antimicrobial peptides that are abundant in the skin secretions of the arboreal frogs Phyllomedusa bicolor and P. sauvagii. In vitro, these peptides are microbicidal against a wide variety of microorganisms including Gram-positive and Gram-negative bacteria, yeasts, protozoa and fungi. To date, 6 dermaseptin B mature peptides, 24-34 residues long, 2 dermaseptin B cDNAs and 2 gene sequences have been identified in P. bicolor. To assess dermaseptin related genes further, we screened a P. bicolor genomic library with 32P-labeled cDNAs coding either for prepro-dermaseptins B1 or B2 (adenoregulin). A gene sequence was identified that coded a novel dermaseptin B, termed Drg3, which exhibits 23-42% amino acids identities with other members of the family. Analysis of the cDNAs coding precursors for several opioid and antimicrobial peptides originating from the skin of various amphibian species revealed that the 25-residue preproregion of these preproforms are all encoded by conserved nucleotides encompassed by the first coding exon of the Drg3 gene. Synthetic dermaseptin Drg3 exhibited a bactericidal activity towards several species of mollicutes (wall-less eubacteria), firmicutes (Gram-positive eubacteria), and gracilicutes (Gram-negative eubacteria), with minimal inhibitory concentrations (MICs) ranging from 6.25 to 100 microM. Experiments performed on Acholeplasma laidlawii cells revealed that this peptide is membranotropic and that if efficiently depolarizes the plasma membrane.

Processing of thyrotropin-releasing hormone (TRH) prohormone in the rat olfactory bulb generates novel TRH-related peptides.

Based on the deduced amino acid sequence of rat TRH prohormone (pro-TRH), proteolytic processing of this polyprotein precursor is expected to produce, beside TRH, several other novel peptides. These peptides should correspond to connecting segments flanking the repeated TRH progenitor sequence and to various C- and/or N-terminally extended forms of TRH. The profile of the endogenous products of the TRH system was studied in rat brain using multiple RIAs coupled to molecular sieve filtration and HPLC separations. In extracts from the rat hypothalamus, TRH and two pro-TRH-connecting peptides, prepro-TRH-(160-169) and prepro-TRH-(178-199) were detected in molar ratios corresponding to those expected for a nearly complete processing of the prohormone molecule. In the olfactory bulb, pro-TRH is processed differently, since peptides containing TRH at their N-termini, [pGlu172] prepro-TRH-(172-199) and [pGlu154]prepro-TRH-(154-169), were found to be major end products along with prepro-TRH-(160-169) and prepro-TRH-(178-199). The dissimilarity in tissue content suggests that differential processing of TRH precursor by various enzymatic pathways may act as a regulating mechanism for TRH and TRH-related activities. The cellular localization of C-terminally extended forms of TRH in rat olfactory bulb was examined by the indirect immunoperoxidase method, using antisera directed against prepro-TRH-(160-169) and pre-pro-TRH-(178-199). Cell bodies and nerve fibers were detected in the glomerular and external plexiform layers of the main olfactory bulb. The presence of extended forms of TRH in interneurons and middle tufted cells of the main olfactory bulb suggests that in light of the recent biological properties described for prepro-TRH-(160-169), these peptides may act as neuromodulators for olfactory epithelium inputs or neurotransmitters within more rostrally located olfactory areas in the forebrain.

Isolation of dermenkephalin from amphibian skin, a high-affinity delta-selective opioid heptapeptide containing a D-amino acid residue.

The predicted amino acid sequence of the biosynthetic precursor of dermorphin, a highly potent and nearly specific mu-opioid peptide from amphibian skin, contains four repeats of the dermorphin progenitor sequence and one single copy of a different heptapeptide sequence. We have developed a specific enzyme immunoassay and used synthetic peptides to detect and purify the new predicted heptapeptide (2.4 micrograms/g dry skin) from the skin of the Phyllomedusa sauvagei frog from which dermorphin was originally isolated. The identity of the novel pro-dermorphin related peptide, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2, was established by co-chromatography with synthetic peptides on reverse-phase HPLC, immunological analysis, gas-phase sequencing, mass spectrometry and by pharmacological assays. Opioid-binding assays in vitro demonstrated that both the natural and synthetic heptapeptides displayed exceptionally high selectivity and affinity towards the delta-opioid receptors. Because of its origin and its delta-opioid (enkephalin) activity and specificity, this novel D-amino acid containing peptide is named dermenkephalin.

Modulation of the biological activity of thyrotropin-releasing hormone by alternate processing of pro-TRH.

Thyrotropin-releasing hormone prohormone contains multiple copies of TRH linked together by connecting sequences. Like other plurifunctional prohormone proteins, pro-TRH undergoes differential proteolytic processing in various tissues to generate, beside authentic TRH, several other novel peptides corresponding to C-terminally extended forms of TRH and connecting fragments. The pro-TRH connecting peptides are, together with TRH, predominant storage forms of TRH-precursor related peptides in the rat hypothalamus. Connecting peptides are co-localized with TRH in the median eminence nerve endings and co-released through a mechanism involving voltage-operated Ca2+ channels. The connecting peptide Ps4 is involved in potentiation of the action of TRH on thyrotropin hormone release by pituitary in vitro and in vivo through interactions with a specific pituitary cell receptor coupled to dihydropyridine and omega-connotoxin sensitive Ca2+ channels of the L-type. It also causes dose-dependent increases in the steady state levels of mRNAs of TSH and prolactin through stimulation of the respective gene promoter activities. These findings indicate that Ps4 and TRH, two peptides which originate from a single multifunctional biosynthetic precursor, can function on the same target tissues in a coordinate manner to promote hormonal secretion. This suggests that differential processing of the TRH prohormone may have the potential to modulate the biological activities of TRH.

Opioid activity of dermenkephalin analogues in the guinea-pig myenteric plexus and the hamster vas deferens.

1. To elucidate the structural features required for selective and potent action of dermenkephalin at the delta-opioid receptor, a series of analogues of dermenkephalin and dermorphin were tested for their effectiveness in depressing electrically-evoked contractions of the vas deferens of the hamster (delta-opioid receptors) and the guinea-pig myenteric plexus-longitudinal muscle preparation (mu- and kappa-opioid receptors). 2. Dermenkephalin was more selective and more potent at delta-receptors than the delta-ligand [D-Pen2, D-Pen5]-enkephalin. The responses to dermenkephalin in the hamster vas deferens were increased by addition of peptidase inhibitors; the maximum effect was obtained with 3 microM thiorphan. 3. [L-Met2]-dermenkephalin had 0.2% and [L-Ala2]-dermorphin 0.01% of the agonist activity of the corresponding endogenous peptides which have D-amino acids in position 2. The pharmacological activity of these analogues was unaffected by inhibition of peptidases. This emphasizes the role that the D-configuration plays in determining the bioactive folding of these highly active peptides. 4. Dermenkephalin-(1-6)-NH2 was more potent at delta-receptors than at mu-receptors whereas, dermenkephalin-(1-4)-NH2 is a selective mu-agonist, having no activity at delta-receptors. 5. Substitution of the C-terminal tripeptide of dermorphin with the C-terminal tripeptide of dermenkephalin abolished the mu-receptor preference of dermorphin. The resulting hybrid peptide, Tyr-D-Ala-Phe-Gly-Leu-Met-Asp-NH2 was as potent as dermenkephalin at delta-receptors. A shift towards a preference for delta-receptors was obtained when the C-terminal tetrapeptide of dermorphin was replaced by the C-terminal tetrapeptide of dermenkephalin. 6. Substitution of Asp by Asn in position 7 of dermenkephalin caused an increase in mu-receptor potency and a decrease in delta-receptor potency, resulting in a 20 fold decrease in mu-receptor selectivity. Dermenkephalin-(1-6)-NH2 and [Asn7]-dermenkephalin have almost identical delta-receptor agonist potencies and ratios of IC50 in the myenteric plexus to IC50 in the hamster vas deferens. 7. The results obtained emphasise the importance of a negative charge at the C-terminus of dermenkephalin for selectivity at the delta-opioid receptor. Furthermore, the hydrophobic residues Leu5 and Met6 may be critical in ensuring tight binding to the receptor which results in high agonist potency.

Opioid peptides from frog skin.

The skin of the South American frogs Phyllomedusa secretes, in addition to numerous mammalian-like hormones and neuropeptides, several gene-encoded opioid peptides that contain a D-amino acid in position 2 of their sequence. Dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2, dermenkephalin/deltorphin A, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 and the deltorphins, Tyr-D-Ala-Phe-Xaa-Val-Val-Gly-NH2 (where Xaa is either Asp or Glu) are highly potent at, and exquisitely selective, for the mu- and delta-opioid receptors. D-Ala and D-Met present in dermorphin and related peptides are coded for by the usual codons in the corresponding messenger RNAs. Prepro-dermorphin/dermenkephalin and prepro-deltorphins have considerable sequence identities to precursors encoding 10-46-residue-long antimicrobial peptides--dermaseptins, brevinins, temporins, esculentins and gaegurins--originating from various amphibian species. The similarity between the prepro-regions of precursors encoding end products with strikingly different structures and biological activities supports the suggestion that the genes encoding these peptides are all members of the same family.

The putative immunity protein of the gram-positive bacteria Leuconostoc mesenteroides is preferentially located in the cytoplasm compartment.

Immunity proteins are though to protect bacteriocin-producing bacterial strains against the bactericidal effects of their own bacteriocin. The immunity protein which protects the lactic acid bacterium Leuconostoc mesenteroides against mesentericin Y105(37) bacteriocin was detected and localized by immunofluorescence and electron microscopy, using antibodies directed against the C-terminal end of the predicted immunity protein. The antibodies recognized the immunity proteins of various strains of Leuconostoc, including Leuconostoc mesenteroides and Leuconostoc gelidum. This study demonstrated that immunity proteins produced by Leuconostoc mesenteroides accumulated in the cytoplasmic compartment of the bacteria. This is in contrast with other known immunity proteins, such as the colicin immunity proteins, which are integral membrane proteins possessing three to four transmembrane domains.

Dermorphin and related peptides in rat tissues.

Dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2), a naturally occurring peptide isolated from arboreal frog skin, is endowed with outstanding structural and biological features. It has no structural community with the sequence of mammalian opioid peptides and is a unique example of a D-aminoacid containing peptide which is synthesized via ribosomal route. Dermorphin is the most potent of the opioid peptides or opiates in producing long lasting analgesia and catalepsy. Since most amphibians' secretory peptides have counterparts in the mammalian central nervous system and gastrointestinal tract, we have developed a sensitive enzyme immunoassay that can detect 1 pg dermorphin to verify the possibility of dermorphin or dermorphin-related peptides occurrence in mammalian tissues. Dermorphin-related peptides were purified by fast protein liquid chromatography followed by reverse phase high pressure liquid chromatography. Identification was achieved by chromatographic comparison with synthetic standards and immunological analysis. A peptide behaving like authentic dermorphin was detected (2 ng/g) in rat small intestine. Immunoreactive species of higher Mr were also detected in the brain, adrenal glands and gastrointestinal tract, they may represent extended forms of dermorphin or homologous peptides.

Mechanism of delta-opioid receptor selection by the address domain of dermenkephalin.

Dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-AspNH2) is a highly potent and selective delta-opioid peptide isolated from frog skin. It was recently recognized that the C-terminus His4-Leu5-Met6-Asp7NH2 of dermenkephalin was responsible for the addressing of the peptide towards the delta-opioid receptor. In order to investigate the role played by residues 4, 5 and 6 in this 'delta address', we synthesized and evaluated 20 new analogues for their ability to displace tritiated ligands from mu- and delta-opioid sites. Results showed that position 4 of dermenkephalin contributes to delta selectivity independently of delta-opioid receptor binding by preventing a high affinity mu binding. Position 5 requires a hydrophobic side chain to enhance delta affinity. A high delta affinity was obtained with any amino acids introduced in position 6 suggesting that residue 6 serves as a neutral spacer. Thus, the main features responsible for the high delta-opioid selectivity of dermenkephalin are electrostatic repulsions with the mu-opioid receptor, additional hydrophobic interactions with the delta-opioid receptor and folding of the C-terminal domain.

Mie light-scattering granulometer with adaptive numerical filtering. I. Theory.

A search procedure based on a least-squares method including a regularization scheme constructed from numerical filtering is presented. This method, with the addition of a nephelometer, can be used to determine the particle-size distributions of various scattering media (aerosols, fogs, rocket exhausts, motor plumes) from angular static light-scattering measurements. For retrieval of the distribution function, the experimental data are matched with theoretical patterns derived from Mie theory. The method is numerically investigated with simulated data, and the performance of the inverse procedure is evaluated. The results show that the retrieved distribution function is quite reliable, even for strong levels of noise.

Structural requirements for dermorphin opioid receptor binding.

Structural features influencing binding activity of dermorphin to opioid receptors have been investigated in the rat brain through the synthesis and evaluation of binding affinity of a series of synthetic dermorphin analogs. Tritiated dermorphin was used as primary ligand. The single population of high affinity dermorphin binding sites present in the rat brain is clearly of an opioid nature since bound radiolabeled dermorphin was fully displaced with high affinity either by morphine or naloxone. Displacement of tritiated dermorphin by all alkaloid opiates or dermorphin related peptides tested was monophasic, consistent with simple competitive inhibition at a single population of binding sites. Dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) was the most potent competitor in all experiments. The D-configuration of the amino acid residue in position 2 was found to be of crucial importance for binding. Replacement of D-Ala2 with L-Ala led to a deleterious effect, this analog being 1/5000th as potent as dermorphin in displacing bound tritiated dermorphin from its receptor. Shorter dermorphin homologs, dermorphin-(1-4)-NH2 and dermorphin-(1-3)-NH2, were found to be 20 and 40-fold less potent, respectively, than dermorphin. The C-terminal carboxamide function is of significant importance for manifestation of the full intrinsic binding potency of dermorphin. Deamidated dermorphin had 1/5th the potency of the parent peptide. This suggests that while the whole dermorphin sequence is required for the expression of the full intrinsic binding activity of the molecule, the N-terminal tripeptide is a key structure as it contains the features which allow receptor recognition.

Identification of a D-alanine-containing polypeptide precursor for the peptide opioid, dermorphin.

The naturally occurring amphibian skin peptides dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) and dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) are highly potent and selective agonists at the mu- and the delta-opioid receptors, respectively. For peptides synthesized by animal cells, they have a rather peculiar structural feature of containing a D-amino acid residue in their sequence which imparts biological activity on them. The cloned cDNA encoding the prodermorphin precursor contains the usual alanine and methionine codons at positions where D-alanine and D-methionine are present in the mature products. In this study, dermorphin precursor was characterized in extracts from amphibian skin by antisera recognizing distinct epitopes within the predicted structure of pro-dermorphin. Proteolytic digestion of purified endogenous pro-dermorphin generated a peptide containing a D-alanine in position 2, identified as prepro-dermorphin-(80-89), i.e. Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-Gly-Glu-Ala. In addition, analysis of skin extracts by enzyme immunoassays coupled to high performance liquid chromatography separations revealed the presence of, besides dermenkephalin, novel dermenkephalin-related peptides, i.e. [L-Met2]dermenkephalin, dermenkephalin-OH, and [Met(O)6]dermenkephalin. [L-Met2]dermenkephalin was present in frog skin in a concentration of about 100 times that of dermenkephalin. These observations confirm that, despite the presence of D-amino acid residues, dermorphin and dermenkephalin are genuine products of post-translational processing of a ribosomally made precursor. They suggest that D-Ala and D-Met develop from a dehydrogenation/hydrogenation stereoinversion of their corresponding L isomers incorporated into pro-dermorphin, a process that occurs with low efficiency at an early stage of biosynthesis.

Mie Light-Scattering Granulometer with an Adaptive Numerical Filtering Method. II. Experiments.

A nephelometer is presented that theoretically requires no absolute calibration. This instrument is used for determining the particle-size distribution of various scattering media (aerosols, fogs, rocket exhausts, engine plumes, and the like) from angular static light-scattering measurements. An inverse procedure is used, which consists of a least-squares method and a regularization scheme based on numerical filtering. To retrieve the distribution function one matches the experimental data with theoretical patterns derived from Mie theory. The main principles of the inverse method are briefly presented, and the nephelometer is then described with the associated partial calibration procedure. Finally, the whole granulometer system (inverse method and nephelometer) is validated by comparison of measurements of scattering media with calibrated monodisperse or known size distribution functions.

Dermenkephalin and deltorphin I reveal similarities within ligand-binding domains of mu- and delta-opioid receptors and an additional address subsite on the delta-receptor.

Dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2), dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) and deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) are the first naturally occurring peptides highly potent for and almost specific to the mu- and delta-opioid receptors, respectively. The amino-terminal domains Tyr-D-X-Phe (where X is either Ala or Met) of these peptides behave as selective and potent mu-receptor ligands. Routing of Tyr-D-X-Phe to the delta- or the mu- receptor is associated with the presence or the absence at the C-terminus of an additional hydrophobic and negatively charged tetrapeptide by-passing the mu-addressing ability of the amino-terminal moiety. A study of 20 Tyr-D-X-Phe-Y-NH2 analogs with substitution of X and Y by neutral, hydrophobic, aromatic amino acids as well as by charged amino acid residues shows that tetrapeptides maintain high binding affinity and selectivity for the mu-opioid receptor. Although residue in position 4 serves a delta-address function, the tripeptide motif at the C-terminus of dermenkephalin and deltorphin I are critical components for high selectivity at delta-opioid receptor. Results demonstrate that mu- and delta-opioid receptors share topologically equivalent ligand-binding domains, or ligand-binding sequences similarities, that recognized Tyr-D-X-Phe as a consensus message-binding sequence. The delta-receptor additionally contains a unique address subsite at or near the conserved binding domain that accommodates the C-terminal tetrapeptide motif of dermenkephalin and deltorphin I.

Processing of thyrotropin-releasing hormone prohormone (pro-TRH) generates pro-TRH-connecting peptides. Identification and characterization of prepro-TRH-(160-169) and prepro-TRH-(178-199) in the rat nervous system.

Rat thyrotropin-releasing hormone prohormone (pro-TRH) contains five separate copies of the TRH progenitor sequence: Gln-His-Pro-Gly. Each of the five sequences is flanked by pairs of basic residues and linked together by one of several predicted connecting sequences. Two of the pro-TRH-connecting peptides, prepro-TRH-(160-169) and prepro-TRH-(178-199), were detected in extracts of rat neural tissues by radioimmunoassay using antibodies directed against the corresponding synthetic probes. Endogenous prepro-TRH-(160-169) and prepro-TRH-(178-199) were purified by gel exclusion chromatography, reverse-phase high pressure liquid chromatography, and ion-exchange chromatography. Structural identification of each peptide was achieved by chromatographic comparison with synthetic standards, immunological analysis, and tryptic mapping. Equimolar amounts of these connecting fragments were observed in hypothalamus and spinal cord. Quantification of TRH in spinal cord and hypothalamus extracts revealed the presence of 4.9-6.3 mol of TRH/mol of prepro-TRH-(178-199) and 4.4-6 mol of TRH/mol of prepro-TRH-(160-169), respectively. By using the indirect immunofluorescence technique, prepro-TRH-(178-199) immunoreactive cell bodies were found in the paraventricular nucleus of the hypothalamus, and a dense plexus of immunopositive nerve terminals was observed in the external zone of the median eminence, in a distribution similar to that described for TRH. These studies demonstrate that prepro-TRH-(160-169) and prepro-TRH-(178-199) are, together with TRH, predominant storage forms of the TRH precursor in hypothalamus and spinal cord, being present in molar ratios corresponding to those expected for a nearly complete processing of the prohormone molecule. The presence of pro-TRH-connecting peptides in various brain regions, including the median eminence, suggests that these peptides might act as neuromodulators in the central nervous system and/or neuroendocrine signals at the pituitary level. In the olfactory lobes, prepro-TRH is processed differently since a C-terminally extended form of TRH, prepro-TRH-(172-199), is found as a major end product along with lower but significant amounts of prepro-TRH-(178-199) and prepro-TRH-(160-169). The striking difference in pro-TRH processing patterns among the various tissues examined suggests differential regulating mechanisms for TRH and/or TRH-related activities.

Characterisation and visualisation of [3H]dermorphin binding to mu opioid receptors in the rat brain. Combined high selectivity and affinity in a natural peptide agonist for the morphine (mu) receptor.

Dermorphin, Tyr-DAla-Phe-Gly-Tyr-Pro-Ser-NH2, a potent opioid peptide isolated from amphibian skin, is endowed with outstanding structural and biological features. It has no common structure with mammalian opioid peptides and is a unique example of a peptide, synthesized by an animal cell, which contains a D-amino acid in its native sequence. We have undertaken a complete evaluation of the receptor selectivity of dermorphin, together with the binding characteristics and receptor distribution of [3H]dermorphin in the rat brain. 1. Dermorphin was tested for its relative affinity to mu-, delta- and chi-opioid receptors by determining its potency in displacing the selective mu-receptor ligand [3H]Tyr-DAla-Gly-MePhe-Gly-ol (where Gly-ol = glycinol), the prototypic delta-receptor ligand [3H]Tyr-DPen-Gly-Phe-DPen (where DPen = beta, beta-dimethylcysteine) and the chi ligand [3H]ethylketocyclazocine from rat brain and/or guinea pig cerebellum membrane preparations. Inhibitory constant (Ki) values of dermorphin were 0.7 nM, 62 nM and greater than 5000 nM respectively for mu, delta and chi sites, indicating a selectivity ratio Ki(delta)/Ki(mu) = 88. Under similar conditions, Tyr-DAla-Gly-MePhe-Gly-ol, which is regarded as one of the most selective high-affinity mu-agonist available, exhibited a selectivity ratio of 84. 2. Specific binding properties of tritium-labeled dermorphin (52 Ci/mmol) were characterized in the rat brain. Equilibrium measurements performed over a large range of concentrations revealed a single homogeneous population of high-affinity binding sites (Kd = 0.46 nM; Bmax = 92 fmol/mg membrane protein). 3. Profound differences were observed in the potencies displayed by various selective opiates and opioids ligands in inhibiting the specific binding of [3H]dermorphin. The rank order of potency was in good agreement with that obtained with other mu-selective radiolabeled ligands. 4. Receptor autoradiography in vitro was used to visualize the distribution of [3H]dermorphin binding sites in rat brain. The labeling pattern paralleled that observed using other mu probes. Binding parameters and selectivity profile of [3H]dermorphin on slide-mounted sections were similar to those obtained with membrane homogenates. 5. Finally, intracerebroventricular administration of synthetic dermorphin into mice showed that this peptide is the most potent analgesic known to date, being up to 5 and 670 times more active than beta-endorphin and morphine, respectively. Higher doses induced catalepsy. The overall data collected demonstrate that dermorphin is the first among the naturally occurring peptides to be highly potent and nearly specific super-agonist towards the morphine (mu) receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of the receptor binding profile of (3H)-dermorphin in the rat brain.

Amphibian skin synthesizes a variety of biologically active peptides. Of these, dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) is an extraordinarily potent opioid peptide up to 1000 times more active than morphine in inducing analgesia after intracerebroventricular administration. Dermorphin has little in common with the sequence of all hitherto known mammalian opioid peptides and is unique in having a D-amino acid residue in position 2. Specific binding properties of tritium labeled dermorphin were characterized in the rat brain. Scatchard or Hill analysis of equilibrium measurements performed over a large range of concentrations revealed a single population of dermorphin binding sites with a Kd value of 0.46 nM. Dermorphin and the selective mu-receptor ligand (D-Ala2, MePhe4, Gly5-ol)-enkephalin (DAGO) had similar high potencies in competing with (3H)-dermorphin binding, whereas the inverse holds for the prototypical delta receptor ligand (D-Pen2, D-Pen5)-enkephalin (DPDPE), which exhibited a potency three orders of magnitude lower. Dermorphin was tested for its relative affinity to mu and delta binding sites by determining its potency in displacing (3H)-DAGO and (3H)-DPDPE from rat brain membrane preparations. Based on these comparisons, dermorphin exhibited a selectivity ratio Ki(DPDPE)/Ki(DAGO) = 100, a value almost identical to that of DAGO, this ligand being considered as the protypical mu-receptor probe. The high affinity and selectivity of (3H)-dermorphin together with its very low nonspecific binding make this peptide a useful tool for dissecting the role(s) of the mu-receptor(s).

Solid phase synthesis of somatostatin-28 II. A new biologically active octacosapeptide from anglerfish pancreatic islets.

Somatostatin-28 II, an octacosapeptide recently isolated from anglerfish pancreatic islets, was synthetized by the solid phase method along with its somatostatin-14 II and somatostatin-28 II-(1-12) corresponding domains. Homogeneity of the synthetic peptides was demonstrated by analytical RP-HPLC, thin layer chromatography and electrophoresis. The peptides were further characterized by amino acids analysis, fast atomic bombarding mass spectrometry and/or 252Cf plasma desorption mass spectrometry. Synthetic somatostatin-28 II and somatostatin-14 II displace equally well the potent agonist (Tyr0,D-Trp8)-somatostatin-14 from its specific binding sites on anterior pituitary cells membranes. Both peptides activate adenylate cyclase from dispersed rat anterior pituitary cells.

Isolation and amino acid sequence of the TRH-potentiating peptide from bovine hypothalamus.

A neuropeptide termed TRH-potentiating peptide, which potentiates TRH-evoked thyrotropin secretion by antehypophysis in vitro, was isolated from an acetonic powder of bovine hypothalamus. The peptide was purified to homogeneity by a 3-step protocol involving molecular sieve filtration, ion-exchange chromatography and reverse phase high performance liquid chromatography. The complete amino acid sequence of the decapeptide was determined as Ser-Phe-Pro-Trp-Met-Glu-Ser-Asp-Val-Thr by automated Edman degradation with a solid-phase sequencer. Bovine TRH-potentiating peptide is structurally identical to Ps4, a decapeptide which was deduced from the cDNA encoding the rat TRH precursor. This study provides for the first time a direct chemical evidence for the existence of non-TRH peptides originating from posttranslational processing of the TRH precursor in vivo.

The aspartic acid in deltorphin I and dermenkephalin promotes targeting to delta-opioid receptor independently of receptor binding.

Recent studies on the highly potent and selective delta-opioid agonists demenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) and deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) suggested that key structural features necessary for specific targetting to the delta-opioid receptor are located within the C-terminal halves of these naturally occurring heptapeptides. To investigate the contribution of aspartic acid 4 residue in deltorphin I and aspartic acid 7 residue in dermenkephalin to the delta-addressing ability of the C-terminal ends, fourteen analogs were synthesized and assessed for their ability to bind to mu and delta-opioid receptors in rat brain membrane homogenates. Results showed that i/ although the tetrapeptide C-terminus of dermenkephalin and deltorphin I differ in amino acid composition, they play a similar role in specifying correct addressing of these peptides to the delta-receptor, ii/ the negatively charged side chain of aspartic acid 4 residue in deltorphin I and aspartic acid 7 residue in dermenkephalin is not involved in binding contact at the delta-receptor site, nor in maintaining a delta-bioactive folding of the peptides, iii/ these side chains are, in contrast, functionally or structurally required to confer high delta-selectivity by preventing mu-site recognition and/or binding.